PROJECT SUMMARY Chronic opioid use among people living with HIV (PLHIV) is an on-going public health crisis. The interplay between opioids and HIV may accelerate HIV-associated immune dysregulation, chronic inflammation and coagulopathy, and predispose to AIDS and non-AIDS related mortality and morbidities. Published data from in vitro and animal models demonstrate that, in addition to their interactions with opioid receptors, opioids directly bind and activate Toll-like Receptor-4 (TLR-4), promote intestinal damage and microbial translocation, interfere with regulation of lipopolysaccharide (LPS)-induced inflammation, and facilitate HIV replication. Our own preliminary data support that opioid use is associated with advanced immune activation and dysregulated responses to LPS among PLHIV. We hypothesize that opioid exposure disrupts homeostatic anti-inflammatory miRNAs that normally limit TLR-4 mediated monocyte activation in response to LPS, leading to chronic innate immune activation and dysfunction. We propose that opioid exposure will be associated with evidence of advanced intestinal permeability, HIV-associated T cell activation and exhaustion, coagulopathy, and compromised antiviral capacity. In this proposal, we will capitalize on our access to longitudinal clinical samples from PLHIV who also suffer from opioid-use disorders (HIV+/OUD+), who are participating in an independent phase IIb randomized clinical trial (CTN-067) to determine optimal management of combined HIV infection and OUD. Trial participants will receive antiretroviral therapy (ART) plus 6 months of OUD therapy with either 1) the long-acting opioid-antagonist extended release naltrexone (XR-NTX); or 2) treatment-as- usual (TAU) with opioid-agonists. Based on published findings from in vitro and animal models, we propose that XR-NTX will be associated with significant reductions in gut permeability, immune activation and exhaustion, coagulopathy, and gains in T cell anti-viral capacity (Aim 1); will restore miRNAs that normally regulate LPS-induced immune activation (Aim 2); and will promote early viral control and reduction of the inducible HIV reservoir (Aim 3). To address these aims, we will use samples collected from CTN-067 trial participants (N=350), as well as a reference cohort of PLHIV with no history of substance dependency or opioid exposure (HIV+/OUD-; N=100). We will employ advanced techniques including: an ultrasensitive electrochemiluminescence-based platform to measure plasma cytokines; next generation sequencing (NGS) of miRNA and mRNA from resting and LPS-stimulated monocytes; a Tat/rev Induced Limiting Dilution Assay (TILDA) to quantify seeding of the inducible provirus reservoir; and a novel flow cytometry assay to quantify CD4+ T cell subsets that contain provirus expressing the gag protein and new budding cell surface virions. We hope to identify an OUD treatment strategy that simultaneously ameliorates immune dysregulation and promotes viral control; if successful, our findings would provide an innovative pathway to disease management in PLHIV both with and without OUD.